Structural analysis of a novel anionic polysaccharide from Porphyromonas gingivalis strain W50 related to Arg‐gingipain glycans

Abstract

SummaryThe Arg-gingipains (RgpsA and B) of Porphyromonas gingivalis are a family of extracellular cysteine proteases and are important virulence determinants of this periodontal bacterium. A monoclonal antibody, MAb1B5, which recognizes an epitope on glycosylated monomeric RgpAs also cross-reacts with a cellsurface polysaccharide of P. gingivalis W50 suggesting that the maturation pathway of the Arg-gingipains may be linked to the biosynthesis of a surface carbohydrate. We report the purification and structural characterization of the cross-reacting anionic polysaccharide (APS), which is distinct from both the lipopolysaccharide and serotype capsule polysaccharide of P. gingivalis W50. The structure of APS was determined by 1D and 2D NMR spectroscopy and methylation analysis, which showed it to be a phosphorylated branched mannan. The backbone is built up of α α α α -1,6-linked mannose residues and the sidechains contain α α α α -1,2-linked mannose oligosaccharides of different lengths (one to two sugar residues) attached to the backbone via 1,2-linkage. One of the side-chains in the repeating unit contains Man α α α α 1-2Man α α α α 1-phosphate linked via phosphorus to a backbone mannose at position 2. De-O -phosphorylation of APS abolished cross-reactivity suggesting that Man α α α α 1-2Man α α α α 1-phosphate fragment forms part of the epitope recognized by MAb1B5. This phosphorylated branched mannan represents a novel polysaccharide that is immunologically related to the post-translational additions of Arg-gingipains.

Selective Sorting of Cargo Proteins into Bacterial Membrane Vesicles

“…We reasoned that a flippase-deficient strain would be useful to determine whether P. gingivalis O antigen chains contribute to OMV formation. The porR locus is involved in A-LPS biosynthesis (15,26); however, not all of the genes in that locus have been characterized (supplemental Fig. S1).…”

“…LPS is composed of lipid A, a core oligosaccharide, and a long polysaccharide chain named O antigen or O polysaccharide. The human oral pathogen Porphyromonas gingivalis, a major causative agent of chronic periodontitis, produces two classes of LPS, carrying either neutral (O-LPS) (13) or negatively charged (A-LPS) O antigen chains (14,15). In other bacterial species containing both neutral and negatively charged O polysaccharides, only the latter has been found in the OMV.…”

Importance of heterogeneity in Porhyromonas gingivalis lipopolysaccharide lipid A in tissue specific inflammatory signalling

“…Genesis of these factors may share common pathways as suggested previously, in respect to µ-oxo-bisheme, responsible for the black pigment. Since a monoclonal antibody (MAb1B5) raised against the Arg-gingipain RgpA cross-reacted with a cell-surface polysaccharide of P. gingivalis strain W50 (A-LPS) [24,63], it was suggested that the maturation pathway of the Arg-gingipains could be linked to the biosynthesis of a surface carbohydrate [24]. The cross-reacting APS, different from the LPS and serotype capsule polysaccharide, was found to be a phosphorylated branch of mannan backbone.…”

“…This lead to the proposal that the Manα1–2Manα1-phosphate fragment constitutes part of the epitope recognized by MAb1B5. This phosphorylated branched mannan represented a new polysaccharide that appears immunologically connected to the post-translational additions of Arg-gingipains [24,63]. Accordingly, there seem to be common steps in the biosynthesis of A-LPS and the glycosylation of Arg gingipains in P. gingivalis .…”

“…P. gingivalis has two major forms of LPS, O-LPS (Figure 1(a)) [23] and A-LPS (Figure 1(b)) [24–26]. O-LPS is a conventional O -antigen polysaccharide found in most bacteria with Gram-negative characteristics, and A-LPS is an anionic polysaccharide (APS) (Figure 1(b)).…”